Sleep is ubiquitous[1] and vital for life[2], while sleep loss leads to several detrimental effects[3]. However, as demonstrated by the absence of sleep-specific drugs, the mechanism of sleep regulation is unknown. Studies in Drosophila (i.e. fruit flies) show that mutations in genes encoding the Shaker K+ channel[4] and two Shaker channel modulators (Hyperkinetic[5] and Sleepless[6]) dramatically reduce daily sleep time, which is significant because K+ channels seem to have a similar role in the regulation of mammalian sleep as well[7-8]. This proposal includes three lines of studies to investigate the relationships between Shaker, its modulators, and sleep. The first aim is to identify the neuronal populations responsible for the Shaker-dependent regulation of sleep in Drosophila. This will be accomplished by ectopically expressing dominant negative Shaker subunits in select brain areas (via the bipartite GAL4 system) and assaying for abnormal sleep phenotypes. The anticipated results will contribute to our understanding of the neuroanatomy of sleep in Drosophila and allow for future molecular dissection of the sleep regulatory mechanism(s) in these candidate cells. The second aim is to explore the possible cellular mechanism by which the aforementioned sleep- reducing alleles are responsible for their abnormal sleep phenotypes. This will be accomplished by correlating the effects of each of the alleles on synaptic activity and cellular ionic currents recorded at the Drosophila neuromuscular junction with the severities of sleep loss that they induce. The third aim is to determine whether the mechanism by which Sleepless modulates Shaker channel activity is through a direct interaction at the plasma membrane. Identification of such a mechanism would not only provide insight into sleep regulation, but, because this would be a completely novel means by which to specifically enhance Shaker channel activity, could have significant non-sleep implications (e.g. the development of new anti-hypertensive or antiarrythmic agents). PUBLIC HEALTH RELEVANCE: The majority of Americans do not sleep for the recommended "8 hours a night"[9], resulting in sleep deprivation that incurs an estimated ~$15-75 billion per year strain[10-11] on the US economy and is linked to several major health problems (e.g. diabetes, heart disease, cancer)[12-14]. However, we know very little about the biological mechanism of sleep regulation, which could be useful to develop interventions to manipulate our need for sleep. The proposed studies will investigate the brain anatomy and cellular activity that play a role in regulating sleep in the fruit fly, laying the foundation for future studies in mammals.